The polymerase chain reaction (PCR) is based on repeated cycles of denaturation of double stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (eg see Mullis et al U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). The oligonucleotide primers used in PCR are designed to anneal to opposite strands of the DNA, and are positioned so that the DNA polymerase-catalysed extension product of one primer can serve as a template strand for the other primer. The PCR amplification process results in the exponential increase of discrete DNA the length of which is defined by the 5′ ends of the oligonucleotide primers.
In our copending International application entitled “Nucleic acid amplification” filed on 25 Feb. 2003, the entire disclosure of which is incorporated herein by reference, we describe the of method for the selective amplification of a nucleic acid using a primer that includes a region the is an inverted repeat of a sequence in a non-target nucleic acid.
Specificity in PCR amplification of DNA is principally determined by the sequence of the primers in combination with the temperature at which the annealing step is conducted. For closely related sequences, additional approaches have been incorporated to provide selective amplification. Where a sequence difference corresponds to a restriction enzyme site, restriction enzyme digests can be used to cut an unwanted sequence and prevent its amplification. Another method of suppressing amplification is the use of oligonucleotides or PNA (peptide nucleic acid) molecules that anneal to one of the DNA strands, within the region to be amplified and/or overlapping the binding site of one of the primers, and prevent initiation or elongation of DNA synthesis. Such oligonucleotides are designed to preferentially anneal with and suppress amplification of one of two related sequences.
We describe below a novel method for selectively suppressing the amplification of one or more closely related sequences while using PCR primers that can prime and extend on both the target and suppressed sequences.